Replication timing of genes and middle repetitive sequences.

DNA replication in mammals is temporally bimodal. "Housekeeping" genes, which are active in all cells, replicate during the first half of the S phase of cell growth. Tissue-specific genes replicate early in those cells in which they are potentially expressed, and they usually replicate late in tissues in which they are not expressed. Replication during the first half of the S phase is, therefore, a necessary but not sufficient condition for gene transcription. A change in the replication timing of a tissue-specific gene appears to reflect the commitment of that gene to transcriptional competence or to quiescence during ontogeny. Most families of middle repetitive sequences replicate either early or late. These data are consistent with a model in which two functionally distinct genomes coexist in the nucleus.

Hemolytic activity of adenylate cyclase toxin from Bordetella pertussis.

Adenylate cyclase (AC) toxin from B. pertussis enters eukaryotic cells where it produces supraphysiologic levels of cAMP. Purification of AC toxin activity [(1989) J. Biol. Chem. 264, 19279] results in increasing potency of hemolytic activity and electroelution of the 216-kDa holotoxin yields a single protein with AC enzymatic, toxin and hemolytic activities. AC toxin and E. coli hemolysin, which have DNA sequence homology [(1988) EMBO J. 7, 3997] are immunologically cross-reactive. The time courses of hemolysis elicited by the two molecules are strikingly different, however, with AC toxin eliciting cAMP accumulation with rapid onset, but hemolysis with a lag of greater than or equal to 45 min. Finally, osmotic protection experiments indicate that the size of the putative pore produced by AC toxin is 3-5-fold smaller than that of E. coli hemolysin.

Enzymatic activity of adenylate cyclase toxin from Bordetella pertussis is not required for hemolysis.

Adenylate cyclase (AC) toxin from Bordetella pertussis enters cells to cause supraphysiologic increases in cAMP. AC toxin is also hemolytic. Substitution of Lys-58 with a methionine residue by site-directed mutagenesis of the structural gene for AC toxin, cyaA, and introduction of this mutation onto the B. pertussis chromosome results in an organism that synthesizes an enzyme-deficient AC toxin molecule. This mutant toxin molecule exhibits 1000-fold reduction in enzymatic activity relative to wild-type and has no toxin activity in J774 cells. The enzyme-deficient toxin molecule is not, however, impaired in its ability to lyse sheep red blood cells. In order to ascertain the importance of these two separate activities of AC toxin in vivo the enzyme-deficient organisms were used to infect infant mice. The hemolytic, enzyme-deficient mutant organisms are reduced in virulence relative to wild-type organisms after intranasal challenge indicating that, although the enzymatic activity of AC toxin does not contribute to hemolysis, it is this property of the toxin which is important for virulence of B. pertussis.

Structural and functional properties of a 69-kilodalton outer membrane protein of Bordetella pertussis.

A-69-kDa outer membrane protein present on virulent Bordetella pertussis cells is recognized by the agglutinating monoclonal antibodies BPE3, BPD8, and BPE8. The amino acid composition of this protein, purified from heat extracts of B. pertussis BP353 cells, is different from that of the two major fimbrial antigens of B. pertussis, which is consistent with its being a nonfimbrial protein based on other criteria. Western blot analysis using the monoclonal antibody BPE3 demonstrated that a slightly larger but antigenically cross-reactive protein is also expressed by Bordetella bronchiseptica and Bordetella parapertussis. In addition, a large molecular weight species of about 180-kDa is found in outer membrane extracts of B. bronchiseptica which may represent a precursor form of the protein or indicate that the protein can exist as an oligomer. The monoclonal antibody BPD8 directed against the 69-kDa protein almost completely inhibited the enzymatic activity of adenylate cyclase purified from B. pertussis and also inhibited the intoxication of mammalian cells by this enzyme. Since little enzymatic activity was found associated with the purified 69-kDa protein, these data suggest a role for the 69-kDa protein in regulating the adenylate cyclase toxin of B. pertussis. An additional monoclonal antibody directed against the 69-kDa protein, BPE8, decreases lymphocytosis and delays death in mice receiving a respiratory challenge of virulent B. pertussis cells. These studies suggest that further investigation into the role of this protein as a protective antigen and vaccine candidate is warranted.

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution.

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.

The effects of managed behavioral health care utilization review on hospital substance abuse detoxification.

Four groups drawn from patients in a hospital substance abuse detoxification unit were compared to determine if staff training on the basics of managed care would impact the length of the hospital stay authorized by private health insurance providers. Pre- and post-intervention groups were drawn from patients carrying the most frequently used insurance, Blue Cross/Blue Shield (BC/BS) and from patients of all other private insurance providers. Results indicate that the most frequently used provider was authorizing lengths of stay consistent with those of other private providers. However, results also indicated that significantly shorter lengths of stay were being authorized by all of the private insurance providers during the post-intervention period of the study. This study (1) confirms clinical observations showing a trend toward shorter lengths of hospital stays for patients with substance use disorders and (2) confirms that these decisions are being made primarily by insurance providers.

Adenylate cyclase toxin from Bordetella pertussis produces ion conductance across artificial lipid bilayers in a calcium- and polarity-dependent manner.

Adenylate cyclase toxin (AC toxin) from Bordetella pertussis enters target cells to produce supraphysiologic levels of cAMP and, by a cAMP-independent process, is hemolytic. In the present study, we show for the first time that this toxin also produces ion-permeable, cation-selective pores in phospholipid bilayers. The resulting membrane conductance is absolutely calcium-dependent, as are the intoxication and hemolytic activities. It is strongly affected by the polarity and magnitude of the membrane potential and enhanced by the presence of negatively charged phospholipid. AC toxins from two mutants, BPDE386 and BPD377, which are defective in toxin activity, produce little or no conductance. Finally, evaluation of the current-voltage relationships and the concentration dependence of pore formation and of hemolysis reveal a greater than 3rd power dependence, suggesting that a multimer of AC toxin, probably consisting of three or more holotoxin molecules, is involved in pore formation.

Medical complications of anorexia nervosa.

Anorexia nervosa is often characterized by progressive deterioration in many different organ systems. Most medical complications are the result of starvation and can be reversed with a well-planned refeeding program. While some of the complications of anorexia nervosa are predictable physiologic adaptations to the self-imposed starvation, many others are potentially life threatening. It is therefore incumbent upon all primary care physicians to become familiar with this disorder, because it is increasing in incidence and is commonly burdened by substantial chronicity and recidivism.

Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis.

Adenylate cyclase (AC) toxin is present on the surface of Bordetella pertussis organisms and their addition to eukaryotic cells results in increases in intracellular cAMP. To test the hypothesis that surface-bound toxin is the source for intoxication of cells when incubated with B. pertussis, we characterized the requirements of intoxication from intact bacteria and found that this process is calcium-dependent and blocked by monoclonal antibody to AC toxin or antibody against CD11b, a surface glycoprotein receptor for the toxin. Increases in intracellular cAMP correlate with the number of adherent bacteria, not the total number present in the medium, suggesting that interaction of bacteria with target cells is important for efficient delivery of AC toxin. A filamentous haemagglutinin-deficient mutant (BP353) and a clinical isolate (GMT1), both of which have a marked reduction in AC toxin on their surface, and wild-type B. pertussis (BP338) from which surface AC toxin has been removed by trypsin, were fully competent for intoxicating target cells, demonstrating that surface-bound AC toxin is not responsible for intoxication. B. pertussis killed by gentamicin or gamma irradiation were unable to intoxicate, illustrating that toxin delivery requires viable bacteria. Furthermore, CCCP, a protonophore that disrupts the proton gradient necessary for the secretion of related RTX toxins, blocked intoxication by whole bacteria. These data establish that delivery of this toxin by intact B. pertussis is not dependent on the surface-associated AC toxin, but requires close association of live bacteria with target cells and the active secretion of AC toxin.

Adenylate cyclase toxin from Bordetella pertussis: current concepts and problems in the study of toxin functions.

Adenylate cyclase (AC) toxin produced by Bordella pertussis and other Bordella species is a virulence factor and protective antigen with novel properties and activities, which make it attractive as a prototype toxin for study of membrane insertion and delivery to the target cell interior. It is unique among RTX toxins in that it possesses enzymatic (adenylate cyclase) activity, as well as the capacity to create an ion-permeable pore in target cell membranes and lyse erythrocytes. The current issues in understanding AC toxin, which will be discussed here, include the role of acylation in its various activities and the relationship among those several toxin functions.

Microenzymatic fluorescence assay for serum cholesterol.

An enzymatic assay using fluorometric detection for cholesterol determination in serum is described. Results were compared to a conventional enzymatic colorimetric procedure and to the definitive method, which is based on isotope dilution mass spectrometry. Fluorescence detection enhances sensitivity over current colorimetric methods by approximately two orders of magnitude, and the assay response is linear over three orders of magnitude of cholesterol concentration. The reaction is performed in a single step and can be performed with small sample (1 microliter) and reaction (200 microliters) volumes. The fluorescence intensity is stable after a 30-min sample incubation at room temperature. The sensitivity of this fluorescence assay makes it possible to measure subnanomoles of cholesterol, allowing accurate measurement of total cholesterol in 1 microliter of serum or less. This level of sensitivity will also allow measurement of cholesterol in various isolated lipoprotein fractions.

Membrane depolarization prevents cell invasion by Bordetella pertussis adenylate cyclase toxin.

Adenylate cyclase toxin from Bordetella pertussis is a 177-kDa calmodulin-activated enzyme that has the ability to enter eukaryotic cells and convert endogenous ATP into cAMP. Little is known, however, about the mechanism of cell entry. We now demonstrate that intoxication of cardiac myocytes by adenylate cyclase toxin is driven and controlled by the electrical potential across the plasma membrane. The steepness of the voltage dependence of intoxication is comparable with that previously observed for the activation of K+ and Na+ channels of excitable membranes. The voltage-sensitive process is downstream from toxin binding to the cell surface and appears to correspond to the translocation of the catalytic domain across the membrane.

Characterization of binding of adenylate cyclase toxin to target cells by flow cytometry.

Adenylate cyclase (AC) toxin from Bordetella pertussis intoxicates eukaryotic cells by increasing intracellular cyclic AMP (cAMP) levels. In addition, insertion of AC toxin into the plasma membrane causes efflux of intracellular K(+) and, in a related process, hemolysis of sheep erythrocytes. Although intoxication, K(+) efflux, and hemolysis have been thoroughly investigated, there is little information on the nature of the interaction of this toxin with intact target cells. Using flow cytometry, we observe that binding of AC toxin to sheep erythrocytes and Jurkat T lymphocytes is dependent on posttranslational acylation of the toxin. Extracellular calcium is also necessary, with a steep calcium concentration dependence similar to that required for intoxication and hemolysis. Binding of AC toxin is concentration dependent but unsaturable up to 50 micrograms/ml, suggesting that if there is a specific receptor molecule with which the toxin interacts, it is not limiting. Visualization of cells by fluorescence microscopy supports the data obtained by flow cytometry and reveals a peripheral pattern of toxin distribution. AC toxin binds to erythrocytes at both 0 and 37 degrees C; however, the total binding at 0 degrees C is less than that at 37 degrees C. In human erythrocytes, AC toxin does not cause an increase in K(+) efflux or hemolysis. While AC toxin exhibits reduced potency to increase cAMP in these cells than in sheep erythrocytes, there is only a modest reduction in the binding of the toxin as measured by flow cytometry. Further use of this technique will provide new approaches for dynamic and functional analysis of the early steps involved in intoxication, K(+) efflux, and hemolysis produced by AC toxin.

Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin.

Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.

Pertussis toxin effects on T lymphocytes are mediated through CD3 and not by pertussis toxin catalyzed modification of a G protein.

Pertussis toxin (PT) has been shown to have a variety of effects on T lymphocyte function, and its activity has been used to suggest the involvement of a G protein in the early events of T lymphocyte activation. In this report, the effects of PT on T lymphocytes have been investigated in detail. PT at a concentration of 10 micrograms/ml rapidly stimulated early events that are normally induced by occupancy of the TCR complex in Jurkat cells and cloned, murine CTL including increased intracellular Ca2+ concentration, serine esterase release, and induction of Ag non-specific target cell lysis. However, 1-h treatment with this concentration of PT induced a state that was refractory to further receptor stimulation in Jurkat cells but not cloned CTL although substrate membrane proteins were modified to a similar extent in both cell lines. The functional effects of PT were mimicked by the B oligomer of PT which did not, however, catalyze ADP-ribosylation of membrane proteins. In addition, overnight exposure of Jurkat cells to a lower concentration of PT also modified substrate membrane proteins but did not inhibit receptor stimulation. These findings indicate that PT catalyzed ADP-ribosylation of a G protein does not account for the actions of the toxin on T lymphocytes. Finally, direct stimulation of increased intracellular Ca2+ concentration by PT and the B oligomer only occurred in T lymphocytes expressing CD3. This suggests that the mitogenic effect of PT holotoxin is mediated by the interaction of the B oligomer with CD3 and that this may account for many of the effects of PT holotoxin both in vivo and in vitro.

Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells.

Adenylate cyclase (AC) toxins produced by Bacillus anthracis and Bordetella pertussis were compared for their ability to interact with and intoxicate Chinese hamster ovary cells. At 30 degrees C, anthrax AC toxin exhibited a lag of 10 min for measurable cAMP accumulation that was not seen with pertussis AC toxin. This finding is consistent with previous data showing inhibition of anthrax AC toxin but not pertussis AC toxin entry by inhibitors of receptor-mediated endocytosis (Gordon, V. M., Leppla, S. H., and Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069). Treatment of target Chinese hamster ovary cells with trypsin or cycloheximide reduced anthrax AC toxin-induced cAMP accumulation by greater than 90%, but was without effect on pertussis AC toxin. In contrast, incubation of the AC toxins with gangliosides prior to addition to target cells inhibited cAMP accumulation by pertussis AC toxin, but not anthrax AC toxin. To evaluate the role of lipids in the interaction of pertussis AC toxin with membranes, multicompartmental liposomes were loaded with a fluorescent marker and exposed to toxin. Pertussis AC toxin elicited marker release in a time- and concentration-dependent manner and required a minimal calcium concentration of 0.2 mM. These data demonstrate that the requirements for intoxication by the AC toxins from B. anthracis and B. pertussis are fundamentally different and provide a perspective for new approaches to study the entry processes.

Adenylate cyclase toxin from Bordetella pertussis. Identification and purification of the holotoxin molecule.

Bordetella pertussis adenylate cyclase (AC) toxin is a calmodulin-activated adenylate cyclase enzyme which has the capacity to enter eukaryotic target cells and catalyze the conversion of endogenous ATP into cyclic AMP. In this work, the AC holotoxin molecule is identified and isolated. It is a single polypeptide of apparent 216 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies which immunoprecipitate AC activity from extracts of wild type B. pertussis (BP338) react with this 216-kDa band on Western blots, and it is absent from a transposon Tn5 mutant (BP348) specifically lacking AC toxin. Isolation of the 216-kDa protein to greater than 85% purity by hydrophobic chromatography, preparative sucrose gradient centrifugation, and affinity chromatography using either calmodulin-Sepharose or monoclonal antibody coupled to Sepharose 4B yields stepwise increases in AC toxin potency, to a maximum of 88.3 mumol of cAMP/mg of target cell protein/mg of toxin. Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities. These data indicate that this protein represents the AC holotoxin molecule.

Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity.

Adenylate cyclase (AC) toxin from Bordetella pertussis interacts with and enters eukaryotic cells to catalyze the production of supraphysiologic levels of cyclic AMP. Although the calmodulin-activated enzymatic activity (ability to convert ATP to cyclic AMP in a cell-free assay) of this molecule is calcium independent, its toxin activity (ability to increase cyclic AMP levels in intact target cells) requires extracellular calcium. Toxin activity as a function of calcium concentration is biphasic, with no intoxication occurring in the absence of calcium, low level intoxication (200-300 pmol of cyclic AMP/mg of Jurkat cell protein) occurring with free calcium concentrations between 100 nM and 100 microM and a 10-fold increase in AC toxin activity at free calcium concentrations above 300 microM. The molecule exhibits a conformational change when free calcium concentrations exceed 100 microM as demonstrated by shift in intrinsic tryptophan fluorescence, an alteration in binding of one anti-AC monoclonal antibody, protection of a fragment from trypsin-mediated proteolysis, and a structural modification as illustrated by electron microscopy. Thus, it appears that an increase in the ambient calcium concentration to a critical point and the ensuing interaction of the toxin with calcium induces a conformational change which is necessary for its insertion into the target cell and for delivery of its catalytic domain to the cell interior.